Multifunctional Photoclick Hyaluronic Acid Nano/Microgels For Efficient Tumor-Targeted Protein Therapy

Malignant tumors have caused increasing amount of morbidity worldwidely.Surgical resection,radiotherapy,and chemotherapy are the most common strategies used to treat cancers in clinic.Chemotherapy was commonly used,while suffers from several drawbacks including poor selectivity,low availability,and serious side effects on normal tissue and organs.Protein therapeutics have received growing interests for treatment of cancers due to potent anticancer effect,high specificity,and low toxicity.However,the clinical applications of protein therapeutics suffer from poor in vivo stability,rapid degradation,and inefficient cellular uptake.Recently,various nanosystems and microsystems have been developed to protect protein from harsh environment,and achieve tumor-targeted protein therapy for cancers.In chapter 1,we have presented a brief introduction on the current status of protein therapeutics for cancers,and summarized the recent work on the development of nanosystems and microsystems for the encapsulation and controlled release of protein therapeutics.In chapter 2,we have designed and developed bioresponsive fluorescent hyaluronic acid nanogels(HA NGs)for facile encapsulation and active tumor-targeting intracellular delivery of apoptotic proteins,including CC and GrB in vivo.HA NGs were readily prepared from two HA derivatives,i.e.HA-cystamine-methacrylate(HA-Cys-MA)and HA-lysine-tetrazole(HA-Lys-Tet),by combing inverse nanoprecipitation and"tetrazole-alkene" photo-click reaction.NGs had a small size of ca.165 nm,narrow PDI of 0.1,negative surface charge(-17.6 mV),excellent stability in serum,and fast protein release under a reductive condition.>80%of CC was released in 10 h under the 10 mM GSH condition,and released CC still maintained its enzymatic bioactivity and secondary structure.CC-and GrB-loaded HA nanogels can effectively target and release proteins to CD44 positive MCF-7 and A549 cancer cells,yielding striking antitumor effects with a half-maximal inhibitory concentration thousands of times lower than those of clinical chemotherapeutics.Remarkably,GrB-NGs at a low dose of 3.8-5.7 nmol of GrB equivalents/kg exhibit complete suppression of tumor growth and minimal adverse effects in nude mice bearing subcutaneous MCF-7 human breast tumor and orthotopic A549 human lung tumor xenografts.This represents a promising nanoplatform for targeted,safe,and efficient delivery of intracellular anticancer protein therapeutics.In order to achieve better tumor selectivity and more efficient target cell uptake of nanogels,in chapter 3,we report on the development of EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels(EGFR/CD44-NGs)to boost protein delivery to ovarian and breast cancers in vitro and in vivo.EGFR/CD44-NGs were obtained via nanoprecipitation and photoclick chemistry from hyaluronic acid derivatives with tetrazole,GE11 peptide/tetrazole and cystamine-methacrylate groups had nearly quantitative loading of therapeutic proteins like cytochrome C(CC)and GrB,a small size of ca.165 nm,excellent stability in serum,and fast protein release under a reductive condition.Flow cytometry assays showed that EGFR/CD44-NGs exhibited over 6-fold better uptake in CD44 and EGFR-positive SKOV-3 ovarian cancer cells than CD44-NGs.In accordance,GrB-loaded EGFR/CD44-NGs(GrB-EGFR/CD44-NGs)displayed enhanced caspase activity and growth inhibition in SKOV-3 cells as compared to GrB-loaded CD44-NGs(GrB-CD44-NGs)control.Intriguingly,the therapeutic studies in SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografted in nude mice revealed that GrB-EGFR/CD44-NGs at a low dose of 3.85 nmol GrB equiv./kg induced nearly complete growth suppression of both tumors,which was obviously more effective than GrB-CD44-NGs,without causing any adverse effects.EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels have appeared as a safe and efficacious platform for cancer protein therapy.Cancer metastatis is regarded as the main cause of cancer-related death.In chapter 4,EGFR/CD44-NGs were employed to load a therapeutic protein(saporin)for the treatment of lung metastasis of 4T1-luc tumors.Flow cytometry assays showed that EGFR/CD44-NGs with GE11/HA molar ratio of 0.96 exhibited over 6.2-fold better uptake than CD44-NGs in 4T1-luc cells.MTT assay showed that Sap-EGFR/CD44-NGs could significantly inhibit the cell proliferation with an IC50 value of 5.4 nM,which was much lower than that of Sap-CD44-NGs(IC50 = 14.4 nM).In vivo anticancer performance showed that Sap-EGFR/CD44-NGs at a low dose of 13.3 nmol Sap equiv./kg caused significant growth inhibition of metastatic nodules in lung site without causing any adverse effects.EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels have appeared as a safe and efficacious platform for metastatic cancer protein therapy.In chapter 5,we have designed and developed homogeneous hyaluronic acid microgels(HMGs)for local sustained delivery of therapeutic proteins like Herceptin to ovarian cancer.HMGs were readily prepared from two HA derivatives,i.e.HA-(2-aminoethyl methacrylate)(HA-AMA)and HA-lysine-dimethylamino tetrazole(HA-Lys-MTet),by combing microfluidics and "tetrazole-alkene" photoclick reaction.HMGs had homogenous sizes,and the size could be well controlled in the range of 25-50μm by changing the flow rate of oil and water phases.HMGs could be degraded in the presence of hyaluronidase,and the degradation rate was accelerated with increasing concentrations of hyaluronidase.Proteins like Herceptin and IgG could be easily encapsulated into HMGs with a loading content up to 27.2 wt.%.In vitro drug release exhibited that Herceptin was release from HMGs in a sustained profile,in which 80.6%of Herceptin was released at a hyaluronidase concentration of 1 U/mL in 10 days.Importantly,the released Herceptin maintained its secondary structure as characterized by circular dichroism(CD)measurement.The therapeutic studies in SKOV-3 human ovarian carcinoma in nude mice revealed that Herceptin-HMGs at a dose of 30 mg Herceptin equiv./kg induced nearly complete growth suppression of tumors,and the tumor inhibition rate(TIR)was 80.4%.Owing to good biocompatibility,controlled degradation behavior,and high performance of anti-tumor efficacy,the hyaluronic acid microgels would have a great potential on local sustained delivery of therapeutic proteins.In chapter 6,we summarize the highlights and significant achievements in my thesis,and give a future perspective on how to transfer these protein nanotherapeutics from bench to clinic.